Philip Wedegaertner, Ph.D.


Associate Professor
Biochemistry & Molecular Biology

G Proteins: Membrane Targeting and Signaling

Telephone
215-503-3137

Office Address
839 BLSB
233 S. 10th St., Philadelphia, PA 19107

Email Address
P_Wedegaertner@mail.jci.tju.edu

 

Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. My laboratory seeks to understand mechanisms of membrane targeting as they relate to signal transduction using the heterotrimeric G proteins (alpha•beta•gamma) as a model system. G proteins act as molecular switches to relay information from activated cell-surface receptors to appropriate effector proteins. The G protein alpha subunit (Galpha) and the gamma subunit of the beta•gamma dimer contain covalently attached lipid molecules that promote tight binding to cellular membranes. We have demonstrated that one of these modifications, palmitoylation, is dynamically regulated to allow reversible movement of Galpha on and off of the plasma membrane. Yet, the underlying mechanisms that target an initially soluble protein to plasma membranes and regulate its membrane localization remain poorly defined. Currently, research in the laboratory is focused on several overlapping areas.

1. Mechanisms of reversible membrane targeting of G alpha and G beta-gamma subunits. In efforts to understand how multiple membrane targeting signals specify proper plasma membrane localization of Galpha, we have embarked on a series of studies to determine the role of G beta•gamma in this process. We have designed and expressed in cultured cells mutant Galpha that are deficient in binding to G beta•gamma. These Galpha fail to arrive at plasma membranes, and they fail to undergo palmitoylation; these experiments have defined a critical role for G beta•gamma in helping to target Galpha to plasma membranes. Interestingly, G beta•gamma appears to exhibit a reciprocal requirement for Galpha; i.e., in order for G beta•gamma to localize to cellular plasma membranes, it must interact with Galpha. Presently, we are trying to understand where in the cell G alpha and G beta•gamma first interact and why they require each other to localize properly at plasma membranes.

In addition to trying to understand the cellular pathways that target newly synthesized G proteins to their correct subcellular location, we are defining the mechanisms underlying movement, or “trafficking”, of G proteins in response to activation. Stimulation of an appropriate receptor by an extracellular agonist in turn activates the G protein. For at least one Galpha, termed alpha s, this activation leads to its translocation from the plasma membrane into the cytoplasm of the cell. Recently, we have demonstrated that the N-terminus of alpha s is critical for allowing this activation-induced trafficking. The N-terminus of alpha s contains a single site for palmitoylation. However, when we engineered alpha s to contain a variety of different membrane targeting signals in its N-terminus to replace the site of palmitoylation, these mutant alpha s proteins remained at the plasma membrane after activation. These results are consistent with a model in which activation-induced depalmitoylation is a key step in regulated and reversible movement of alpha s.

2. Mechanisms of membrane localization of RhoGEFs and characterization of their interactions with G alpha 13. This laboratory is also studying several members of a recently recognized family of proteins called Regulator of G protein Signaling (RGS) proteins. These proteins share in common a 120 amino acid RGS core domain. The function of these RGS domains appears to be to interact with Galpha and “turn-off” signaling and also, to serve as a site for Galpha to bind and “turn-on” signaling through other domains contained in a particular RGS protein. One class of RGS proteins are Rho guanine-nucleotide exchange factors (GEF) that activate the small GTPase Rho. Members of the Rho family play important roles in coordinating actin cytoskeleton rearrangements, promoting cell growth and oncogenic transformation. These RGS-domain containing RhoGEFs appear to provide a direct link between one Galpha, alpha 13, and Rho. We have found that one of these RhoGEFs, called p115rhoGEF, translocates from the cytoplasm of cells to plasma membranes upon activation of alpha 13. The mechanism of this regulated movement of p115rhoGEF appears to be complex, requiring more than just an interaction between alpha 13 and the RGS domain of p115rhoGEF. In contrast, we have found that another RGS domain-containing RhoGEF, termed PDZ-RhoGEF, associates with an actin-containing complex at plasma membranes in the absence of alpha 13 activation. Our goal is to understand the mechanisms of RhoGEF localization, and thus gain insight into signaling by the alpha 13 family of G proteins. In addition, we are defining the determinants of specificity in alpha 13 that allow it to interact with and activate p115-RhoGEF. Mutants of G alpha 13 that are selectively deficient in interacting with RhoGEFs will allow us to define the contribution of Rho activation to the ultimate cell growth and transformation phenotypes induced by G alpha 13.

3. GRKs: Interactions with G alpha q family members and mechanisms of membrane localization. Lastly, this laboratory has developed a collaborative project with the laboratories of Dr. Jeffrey Benovic (Kimmel Cancer Center, TJU), Dr. Rachel Sterne-Marr (Siena College) and Dr. John Tesmer (U.Texas, Austin). We are defining the molecular determinants that allow alpha q to interact with and be regulated by RGS domain-containing G protein-coupled receptor kinases (GRKs). We have determined that interactions between alpha q and GRK2 are unique compared to other studied G alpha/RGS pairs, and we are in the process of defining this interaction further. Another collaborative project with the Benovic laboratory is identifying and characterizing a membrane targeting domain in GRK4, 5, and 6.

Keywords: G proteins, signal transduction, membrane targeting, lipid modification

 

PubMed Link For Wedegaertner P


Selected Publications


Crouthamel, M., Thiyagarajan, M.M., Evanko, D.S, and Wedegaertner, P.B., N-terminal polybasic motifs are required for plasma membrane localization of G±s and G±q,, Cellular Signalling, in press.Iwanicki M.P., Vomastek T., Tilghman R.W., Martin K.H., Banerjee J., Wedegaertner P.B., and Parsons J.T. (2008) FAK, PDZ-RhoGEF and ROCKII cooperate to regulate adhesion movement and trailing-edge retraction in fibroblasts, J. Cell Sci., 121, 895-905.Jiang, X., Benovic, J.L., and Wedegaertner, P.B. (2007) Plasma membrane and nuclear localization of G protein-coupled receptor kinase 6A, Mol. Biol. Cell, 18, 2960-2969.Billington, C.K., Kong, K.C., Bhattacharyya, R., Wedegaertner, P.B., Panettieri, R.A. Jr., Chan, T.O., and Penn R.B. (2005) Cooperative regulation of p70S6 kinase by receptor tyrosine kinases and G protein-coupled receptors augments airway smooth muscle growth, Biochemistry, 44, 14595-14605.Evanko, D.S., Thiyagarajan, M.M., Takida, S., and Wedegaertner, P.B. (2005) Loss of association between activated G alpha q and G beta-gamma disrupts receptor-dependent and receptor-independent signaling, Cellular Signaling, 17, 1218-1228.Cui, J., Holgado-Madruga, M., Su, W., Tsuiki, H., Wedegaertner, P., and Wong, A.J. (2005) Identification of specific domain responsible for JNK2alpha2 autophosphorylation, J. Biol. Chem., 280, 9913-9920.Grabocka, E. and Wedegaertner, P.B. (2005) Functional consequences of G alpha 13 mutations that disrupt interaction with p115RhoGEF, o­ncogene, 24, 2155-2165.Takida, S., Fischer, C.C., and Wedegaertner, P.B. (2005) Palmitoylation and plasma membrane targeting of RGS7 are promoted by alpha o, Mol. Pharm., 67, 132-139.Day, P.W., Tesmer, J.J.G., Sterne-Marr, R., Benovic, J.L., and Wedegaertner, P.B. (2004) Characterization of the GRK2 binding site of G alpha q, J. Biol. Chem., 279, 53643-53652.<--->Day, P.W., Wedegaertner, P.B., and Benovic, J.L., (2004) Analysis of G protein-coupled receptor kinase RGS homology domains, Methods in Enzymology, in press.<--->Thiyagarajan, M.M., Stracquatanio, R.P., Pronin, A.N., Evanko, D.S., Benovic, J.L., and Wedegaertner, P.B. (2004) A predicted amphipathic helix mediates plasma membrane localization of GRK5, J. Biol. Chem., in press.<--->Banerjee, J. and Wedegaertner, P.B. (2004) Identification of a novel sequence in PDZ-RhoGEF that mediates interaction with the actin cytoskeleton, Mol. Biol. Cell, 15, 1760-1775.<--->Day, P.W., Carman, C.V., Sterne-Marr, R., Benovic, J.L., and Wedegaertner, P.B. (2003) Differential interaction of GRK2 with members of the G alpha q family, Biochemistry, 42, 9176-9184.<--->Wedegaertner, P.B. (2003) Reversible Palmitoylation, in Handbook of Cell Signaling vol. 2, Bradshaw, R. and Dennis, E., eds., Academic Press.<--->Takida, S. and Wedegaertner, P.B. (2003)  Heterotrimer formation, together with isoprenylation, is required for plasma membrane targeting of Gβγ, J. Biol. Chem., 278, 17284-17290.<--->Bhattacharyya, R. and Wedegaertner, P.B. (2003) Characterization of Ga13-dependent plasma membrane recruitment of p115RhoGEF, Biochem. J., 371, 709-720.<--->Bhattacharyya, R. and Wedegaertner, P.B. (2003) Mutation of an N-terminal acidic-rich region of p115RhoGEF dissociates a13 binding and a13-promoted plasma membrane recruitment, FEBS Lett., 540, 211-216.<--->Sterne-Marr, R., Tesmer, J.J.G., Day, P.W., Stracquatanio, R.P., Cliente, J.E., O’Connor, K.E., Pronin, A.N., Benovic, J.L., and Wedegaertner, P.B. (2003) GRK2/Gaq/11 interaction: A novel surface on an RGS homology domain for binding Ga subunits, J. Biol. Chem., 278, 6050-6058. <--->Thiyagarajan, M.M., Bigras, E., Van Tol, H.H.M., Hébert, T.E., Evanko, D.S. and Wedegaertner, P.B. (2002) Activation-induced subcellular redistribution of Gas is dependent upon its unique N-terminus, Biochemistry, 41, 9470-9484.<--->Wedegaertner, P.B. (2002) Characterization of subcellular localization and stability of a splice variant of Gai2, BMC Cell Biology, 3, 12.<--->Evanko, D.S., Thiyagarajan, M.M., Siderovski, D.P., and Wedegaertner, P.B. (2001) Gbg isoforms selectively rescue plasma membrane localization and palmitoylation of mutant Gas and Gaq, J. Biol. Chem. 276, 23945-23953. <--->Bhattacharyya, R., and Wedegaertner, P. B. (2000) Ga13 requires palmitoylation for plasma membrane localization, rho-dependent signaling, and promotion of p115rhoGEF membrane binding. J. Biol. Chem. 275, 14992-14999.<--->Evanko, D.S., Thiyagarajan, M.M., and Wedegaertner, P.B. (2000) Interaction with Gbg is required for membrane targeting and palmitoylation of Gas and Gaq, J. Biol. Chem. 275, 1327-1336.<--->Carman, C.V., Parent, J.-L., Day, P.D., Pronin, A.N., Sternweis, P.M., Wedegaertner, P.B., Gilman, A.G., Benovic, J.L., and Kozasa, T. (1999) Selective regulation of Gaq/11 by an RGS domain in the G Protein-coupled receptor kinase, GRK2, J. Biol. Chem. 274, 34483-34492.<--->Wedegaertner, P.B. (1999) Fatty acid acylation of G protein a subunits, in G Proteins: Techniques of Analysis, Manning, D.R., ed., pp. 153-171, CRC Press, Boca Raton, FL.<--->Wedegaertner, P.B. (1998) Lipid modifications and membrane targeting of Ga.  Biological Signals and Receptors, 7, 125-135.<---> 
 

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